4.2. Forward Gene-Specific Primer Extension

In this step, the pool of Forward Gene-Specific Primers with adjoining Anchor 1 sequences (see Procedure Overview generates sense strands of the target amplicons using the cDNA generated in the previous step as a template.

1. Prepare the Forward Gene-Specific Primer Extension Master Mix as follows for all samples plus 5% extra volume of all components as a safeguard against pipetting error:
   

   Forward GS Primer Extension Master Mix Component	Volume per sample, µl
   RT-EXT Buffer	2
   PCR-Grade Water	5.8
   Forward GS Primer Pool*	2
   DNA Polymerase	0.2
   Total	10

Note: There are different Forward GS Primer and Reverse GS Primer pools for each DriverMap Assay. Please refer to the Product Insert for your specific kit to identify the appropriate component in the Kit.

2. Gently vortex the Master Mix, and spin down briefly to collect droplets. Spin down the cDNA plate, remove the seal, then add 10 μl of the Forward GS Primer Extension Master Mix to each reaction well of the plate:
   

   Component	Volume, µl
   cDNA	10
   Forward GS Primer Extension Master Mix	10
   Total	20

3. Mix contents by pipetting 3 times, seal the plate with a new adhesive film, and spin down to collect droplets.

Note: A new adhesive film is required in order to avoid cross-contamination.

4. Load the plate in the thermal cycler, and run the following program:
   

   Temperature	Time
   98°C	1 min
   64°C	30 min
   4°C	∞

5. Prepare Primer Removal Master Mix as follows for all samples plus 5% extra volume of all components as a safeguard against pipetting error:
   

   Primer Removal Master Mix Component	Volume per sample, µl
   RT-EXT Buffer	2.5
   PCR-Grade Water	9.5
   Primer Removal Enzyme	0.5
   Total	12.5

Note: Sufficient Primer Removal Master Mix is prepared above to use at this point in the procedure (5 μl), plus after two other later steps: the Reverse Gene-Specific Primer Extension (5 μl) and when you Quantify and Combine Samples for NGS (2 μl).

6. Spin down, remove the seal from the plate, then add 5 μl of the Primer Removal Master Mix to each reaction well of the plate (keeping the remainder for follow-up steps listed above):
   

   Component	Volume, µl
   Forward GSP Extension products (above)	20
   Primer Removal Master Mix (prepared above)	5
   Total	25

7. Mix contents by pipetting 3 times, seal the plate, and spin down to collect droplets.

8. Load the plate in the thermal cycler, and run the following program:
   

   Temperature	Time
   37°C	30 min
   95°C	5 min
   4°C	∞

Immediately proceed to the next step (Reverse Gene-Specific Primer Extension)